**5. Genotoxicity biomarker: comet assay**

52 New Approaches to the Study of Marine Mammals

**2.3. Sex identification** 

Berubè & Palsboll [24]**.** 

**3. Fibroblast cell cultures** 

replaced every 48 h with fresh medium.

contaminants reported in the Table 1.

**4. Indirect immunofluorescence tecnique** 

RT23), and immediately placed in cell medium.

RT25), sperm whale (PM6), bottlenose dolphin (TurNic) and striped dolphin (RT1 and

Sex determination in cetaceans was carried out by genetic investigations according to

The development of a non-invasive sampling method for obtaining viable tissue samples for cell cultures from skin biopsies of free-ranging and stranded cetaceans was described by Marsili *et al.* [14]. Successful cell cultures were obtained from all the animals. After the biopsy, skin samples were stored in sterile medium MEM Eagle Earle's salts w/L-glutamine and sodium bicarbonate (Mascia Brunelli, Milan, Italy) + 10% gamma irradiated fetal calf serum (Mascia Brunelli) + 1% MEM not essential aminoacids (NEAA) solution 100x (Mascia Brunelli) + 1% Penicillin/Streptomycin 100x (Mascia Brunelli) + 0.1% Amphotericin B 100x (Mascia Brunelli) at room temperature and was processed within 24 h of collection. In the laboratory, each sample was washed with Earle's balanced salt solution (EBSS; Mascia Brunelli) containing antibiotic (Penicillin/Streptomycin 100x [Mascia Brunelli]) and antimycotic (Amphotericin B 100x [Mascia Brunelli]) solutions. All specimens were handled using sterile techniques. Initially, the collected tissue was cut into small pieces with curved surgical scissors, placed in 30-mm Petri dishes and incubated with Trypsin-EDTA solution 1x (Mascia Brunelli) for 15 min at 37°C. The biopsy fragments were washed again and then placed in Falcon 25 flasks, moistened with medium. After 24 h at 37°C in an incubator with 5% CO2, the cultures were covered with 1 ml of medium. Half of the culture medium was

Third generation fibroblast cell cultures were exposed to the different mixtures of

We used immunofluorescence in fibroblast cultures for a qualitative and semi-quantitative analysis of target proteins CYP1A1, CYP2B and MICA. After a first reaction with the primary polyclonal antibodies (goat anti-rabbit cytochrome P450 1A1 and goat anti-rabbit cytochrome P450 2B; Oxford Biochemical Research (Oxford MI, USA); rabbit polyclonal anti-MICA; Abcam), the cells were treated with the respective secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG (H+L) for CYP1A1 and CYP2B; Alexa Fluor 568 rabbit anti-goat IgG (H+L) for MICA; Invitrogen), labelled with red-fluorescent Alexa Fluor dye. Immunofluorescence was quantified with a specially designed Olympus Soft Imaging Systems macro, *DetectIntZ,* which works with the image acquisition, processing and analysis system, *analySIS^B* (Olympus) [15]. The image analysis procedure has the objective of quantifying, with an adimensional index generated for this purpose, the amount of Alexa

Fibroblast cell cultures (third generation) of striped dolphin (RT23) were subjected to this experimental protocol for 4 h. A cell line was exposed to a mixture of benzo(a)pyrene

(1mM) and beta-naphthoflavone (20mM), solubilized in acetone (0.1%), at three doses (Table 1), plus an acetone (0.1%) control. Fibroblast cell cultures were processed for the comet assay after Caliani *et al.* [13], with some modifications. The cells were centrifuged at 1000 g for 10 min, then were embedded in agarose (0.5% low-melting agarose) and layered on conventional slides, predipped in 1% normal melting agarose. The slides were immersed into a freshly made lysis solution (2.5 M NaCl, 10 mM Tris, 0.1 M EDTA, 1% Triton X-100, and 10% DMSO, pH 10) for at least 1 h at 4°C in the dark. The slides were then placed on a horizontal electrophoresis tray previously filled with freshly prepared cold alkaline buffer and left for 20 min to allow DNA unwinding. Electrophoresis was performed at 25 V and 300 mA for 20 min. The DNA migration was evaluated at three different pH conditions (pH 13, pH 12.1, pH 8). Slides were then neutralized in Tris (0.4 M, pH 7.5) for 3x5 min and stained with Sybr safe 1:10.000 in TE (10 mM Tris-HCl pH 7.5 and 500 mM EDTA pH 7.5) buffer. A total of 50 cells per slide were examined under epifluorescence at 40X magnification. The amount of DNA damage was evaluated as the percentage of DNA migrating out of the nucleus using an image analyser (Komet 6.0 Software, Kinetic Imaging Ltd.), connected to a fluorescent microscope (Olympus BX41).

"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of

Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures 55

**Figure 1.** Basal levels of immunofluorescence (AUF/nucleus) of CYP1A1 in fibroblast cells of sperm

The first result of these experiments in nine cetacean species was the detection of the presence of CYP1A1 and CYP2B in fibroblast cells of all species, revealed by immunofluorescence (Figure 2A-B); a higher basal expression of both proteins was found in Risso's dolphin (GGL1) and bottlenose dolphin (TurNic), while fin whale (RT2) and sperm whale (PM6) showed the lowest levels of these proteins. The Risso's dolphin (GGL1) and the sperm whale (PM6) have a very similar diet, consisting mostly of squid. Nevertheless, they have a very different basal expression of the two cytochromes. As for the other species, very high levels of CYP1A1 were present in the Bryde's whale (MBE3). This mysticete sampled in the Sea of Cortez showed CYP1A1 levels more than 20 times greater than the other mysticete studied, the Mediterranean fin whale (RT2). Regarding the levels of contaminants detected in the blubber of different species, the bottlenose dolphin (TurNic), stranded along the coasts of the Mediterranean Sea, had very high levels of organochlorine contaminants in its blubber (DDTs = 77.4 μg/g lipid weight (l.w.); PCBs = 262.6 μg/g l.w.) that are potent inducers of CYP2B. In fact, especially in this specimen this cytochrome appears to be markedly higher than the levels shown by other species (Figure 2B). But the sperm whale (PM6) was also a stranded specimen found on the Italian coasts (Mediterranean Sea) having high values of these xenobiotics in the blubber. It seems therefore that this basal activity is more species-specific than related to the geographical location, diet, toxicological status, etc.

**6.1. Basal levels of CYP1A1 and CYP2B in different species** 

whale treated with pp'DDT and pp'DDE.

in which the animals were found.
